Systems and methods for attenuated total internal reflectance (ATR) spectroscopy

ABSTRACT

Systems and methods for attenuated total reflectance (ATR) spectroscopy. The systems and methods may generally involve differentiating an acquired image in a spectral domain, restricting a spectral range of the acquired image, subtracting an average absorbance from the restricted image data, and applying a principal component analysis to extract significant spectral features from the restricted image data.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority under 35U.S.C. §120 to U.S. patent application Ser. No. 11/912,486 filed on Sep.8, 2008, now U.S. Pat. No. 8,400,711, which is a National StageApplication of PCT/GB2007/001531 filed on Apr. 26, 2007, which claimspriority to United Kingdom Application No. 0608258.0 filed on Apr. 26,2006.

This invention relates to spectroscopy which makes use of attenuatedtotal internal reflection (ATR).

ATR is a technique used in spectroscopy, such as FT-IR spectroscopy, inorder to obtain spectral measurements from samples which are difficultto analyse by other means such as transmission or reflection. Typicallyapparatus for carrying out ATR measurements will comprise a spectrometerto provide wavelength discrimination, an illumination system fordirecting light onto a sample, an ATR optic which provides a sampleplane and a collecting/detecting system which receives light which hasinteracted with the sample. The ATR optic is arranged in such a way asto reflect all incident light from a designated sample plane by means ofthe phenomenon of total internal reflection. Spectral informationconcerning the sample is derived from the interaction of the sample withan evanescent electric field that exists immediately outside thereflecting surface. The absorption of energy from this field attenuatesthe reflection and impresses spectral information on the light beam.

An imaging ATR system can be constructed based upon these principles byarranging to illuminate an area of a sample and by arranging thecollecting system to have imaging properties. Light returning fromspatially distinct regions of the sample is collected on a detector or adetector array such as a one dimensional or two dimensional array ofdetectors and spectral information is thus collected which can becompiled into a spectral image of sample.

An imaging ATR system can be constructed in the form of a reflectancemicroscope such as the Perkin Elmer Spotlight microscope. In such anarrangement light is directed onto and collected from a reflectivesample by means of an imaging optic. An ATR optic for such a system canconveniently comprise a hemispherical plano-convex lens made of a highrefractive index material such as germanium. The optic is arranged sothat the convex spherical surface is directed towards the microscopeoptic with its centre of curvature arranged to be coincident with thefocal plane of the imaging system. The sample is presented to the flatsurface of the ATR.

The microscope includes a moveable stage which has associated motors formoving the stage in x, y and z directions under processor control.Imaging is carried out using a small linear array detector andphysically moving the stage and therefore the crystal/sample combinationlaterally relative to the optical axis of the microscope. As the stageis moved images can be detected by the detector from different parts ofthe sample and in this way a spatial image can be accumulated.

There are a number of requirements and problems which arise in this typeof arrangement. The region of interest of the sample has to beidentified usually visually and placed approximately at the centre ofthe field of view of the microscope. This usually means removing the ATRcrystal since it is usually made of material such as germanium which isopaque to visible light.

The ATR crystal has to be placed with its sample contacting face inintimate contact with sample. This can lead to problems in achieving aninfrared image which is in focus. The sample may move when the crystalis brought into contact with it. Also the crystal may cause defocus byvirtue of its shape. For example if the thickness of the crystal is notprecisely the same as its radius of curvature. The effect is magnifiedbecause the material of the crystal has a high refractive index ofaround 4. Therefore small manufacturing errors can be significant.

The present invention is concerned with improvements in arrangements forATR spectroscopic systems which attempt to overcome these and otherproblems.

According to a first aspect of the present invention there is providedan accessory for a microscope arranged to carry out ATR measurements,said accessory comprising a support which can be mounted on the movablestage of the microscope, a mounting member for mounting an ATR crystalcarried on said support, said mounting member being so mounted andarranged on the support that it can be moved between a position in whicha crystal mounted on the mounting member is aligned with the opticalaxis of the microscope and a position in which the crystal is displacedfrom the optical axis.

The mounting member may comprise an elongate arm pivotally supported atone end on a first guide pin, said arm being pivotal about said pin toallow said movement of the mounting member.

The other end of the aim may have an opening which engages a second pincarried by the support member when the mounting member is located in theposition in which the crystal lies on said optical axis.

The arm may be raised along the axis of the first guide pin so that saidother end moves clear of the second guide pin to allow said pivotalmovement.

A braking mechanism may be associated with said one end of the arm andsaid first guide pin, said braking mechanism being operative to allow acontrolled descent of the arm along the first guide pin when said arm isreturned to its position in which the crystal is aligned with saidoptical axis.

The braking mechanism may comprise a ring which is carried by said armand locates around said first guide pin, said ring having an innerdiameter which is slightly greater than that of said first guide pin,biasing means operative to bias said ring so that a circumferentialportion thereof frictionally engages a surface part of the guide pin,and manually operable means operable to act against said bias means toreduce or release said frictional engagement and thereby allow axialmovement of the ring relative to the guide pin.

The mounting member may be carried in such a way that it can be removedfrom its mounting and inverted by rotation about its longitudinal axisto allow inspection of the sample engaging surface of the crystal, forexample to check that the crystal is not damaged or contaminated.

This aspect of the invention by use of the movable mounting memberallows the crystal to be mounted on the microscope stage such that iscan be removed and subsequently returned accurately and reproductivelyto its original position.

According to a second aspect of the present invention there is providedan accessory for a microscope arranged to carry out ATR measurements,said accessory including a support which can be mounted on the movablestage of the microscope, a mounting member in which is mounted an ATRcrystal, said crystal having a sample contacting area, and aregistration indicium located such that it is fixed relative to thesample contacting area. The crystal may have a generally hemisphericalsurface opposite to said contacting area and said registration indiciummay be located at the apex region of the hemispherical surface. Theindicium may comprise a flat on the hemispherical surface or a mark onthe hemispherical surface.

According to a third aspect of the present invention there is provided amethod of operating a microscope provided with an accessory according tosaid second aspect said microscope including processing means forcontrolling movement of the movable stage and said processing meanshaving recorded therein a predetermined parameter relating to the heightof the ATR crystal, said method comprising initially moving the stage ofthe microscope to bring the registration indicium into focus and movingthe stage by a predetermined vertical distance defined by said parameterin order to bring into focus a sample contacting said sample contactingarea.

According to a fourth aspect of the present invention there is provideda method of calibrating an ATR crystal for use with a microscopeaccording to said second aspect or a method according to said thirdaspect said method comprising a selecting a test sample which exhibitsstrong absorption within the spectral range of the microscope and whichhas a geometry which will produce sharp spatial edges when contacted byan ATR crystal, said method comprising bringing the sample contactingarea of the crystal into contact with the test sample, acquiring aninfra-red image of the test sample at an initial vertical position ofthe crystal, processing the image to extract slope information for saidedges, repeating the process for different vertical positions of thecrystal, identifying the optimum vertical position as that whichexhibits the maximum slope, and deriving a calibration parameter forsaid crystal according to said identified optimum position.

The processing may include, for each vertical position, spectrallyfiltering the acquired image to extract a spatial map of the absorbanceat a wavelength where the test sample absorbs strongly.

The method may include extracting a cross-section which traverses thespatially sharp features of the absorbance map at said wavelength.

The method may include differentiating the cross-section to extract saidslope data, and measuring the maximum slope for a recognisable featurein the image.

The text sample may be a plastic material such as a microembossedpolymer, e.g. Vikuiti brightness enhancing film.

The second, third and fourth aspects of the invention provide a facilitywhich allows determination of the optimum position of the crystal withrespect to the microscope and which can cope with manufacturingtolerances of the crystal. It can position the crystal at the optimumvertical position for achieving a focussed infra-red image in areproducible manner irrespective of sample thickness.

According to a fifth aspect of the present invention there is providedan accessory for a microscope arranged to carry out ATR measurements,said accessory including a support which can be mounted on the movablestage of the microscope, a mounting member carried on said support formounting an ATR crystal, a sample supporting member disposed below thelocation of the ATR crystal, said sample supporting member having asurface upon which a sample can be received, and pressure applying meansdisposed below said sample supporting member for applying a pressure tosaid sample supporting member in the direction of said crystal.

The pressure applying means may include a spherical member through whichpressure is applied to said sample supporting member. This permitslimited tilt of the supporting member in order to accommodate irregularsamples.

The pressure applying means may include spring bias means.

The spring bias means may include a plunger which is urged towards thecrystal by a spring.

The spherical member may be a ball bearing and said plunger contactssaid ball bearing.

The accessory may include a rack and pinion arrangement coupled to saidplunger, said rack being movable manually to effect rotation of thepinion to cause axial movement of the plunger to thereby apply orrelease pressure applied to the sample supporting member.

The pressure which can be applied to the sample supporting member may beadjustable.

The area of the sample supporting member through which the pressureapplying acts is relatively thin to ensure that the point at which thepressure is applied is as close as possible to the crystal.

This aspect of the invention provides a simple and effective means forensuring that the sample is held in good contact with sample contactingsurface of the crystal.

According to a sixth aspect of the present invention there is providedan accessory for a microscope arranged to carry out ATR measurements,said accessory comprising a support which can be fixed to the movablestage of the microscope, a mounting member carried by said support formounting an ATR crystal, a sample supporting member disposed below thelocation of the ATR crystal, said sample supporting member defining asample receiving surface, said sample supporting member being carried onsaid support so that it is movable relative to the ATR crystal anddefines a sub-stage which can be moved relative to the main stage of themicroscope.

The sample supporting member may comprise a flat upper surface and sidesurfaces and the accessory includes location adjusting means foradjusting the position of the sample supporting member on the support.

The location adjusting means may comprise a pair of screws acting tourge the member against a biasing spring.

The screws may be located so as to act along orthogonal directions andsaid spring is arranged to act along a bisector of said directions.

The surface against which each screw acts may be at a small angle to anaxis through the crystal whereby each screw acts to urge the samplesupporting member towards the support.

The aspect of the invention provides a sub-stage which allows theposition of the sample to be adjusted without affecting any previoussetting of the main stage of the microscope.

It will be appreciated that the features of the various aspects of theinvention defined above can be used in any combination thereof.

The invention will be described now by way of example only withparticular reference to the accompanying drawings. In the drawings:

FIG. 1 is a schematic side view illustrating the principal elements of aknown FT-IR microscope;

FIG. 2 is a schematic perspective view of an accessory for an ATRmicroscope, said accessory being constructed in accordance with oneembodiment of the present invention;

FIG. 3 is a perspective view showing the accessory located in themovable stage of a microscope;

FIG. 4 is a section on the line Z-Z of FIG. 2,

FIG. 5 is a section similar to FIG. 3 showing the location of theaccessory in a microscope in relation to the objective cassegrain lens,and

FIG. 6 is a section on a larger scale of part of FIG. 4;

FIG. 7 is a plan view of the accessory;

FIG. 8 is a section on the line V-V of FIG. 7;

FIG. 9 is a section on the line W-W of FIG. 7, and

FIGS. 10 and 11 illustrate the operation of an imaging microscope whichincorporates the accessory.

Referring to FIG. 1 this shows the principal elements of an FT-IRmicroscope and these include an optical microscope 10 which is disposedabove a view/IR mirror 11 which in turn is disposed above a remoteaperture 12. Located below the remote aperture 12 is atransmittance/reflectance mirror 14 which is positioned above anobjective cassegrain assembly 16 and a condenser cassegrain assembly 18.Between the two cassegrains is disposed a movable stage 20 which definesthe position of the sample for analysis. Located below the condensercassegrain 18 there is a flat mirror 22 which can direct radiation froma coupling optic 24 which is in turn configured to receive radiationfrom a radiation source. A microscope of this type can be used for bothreflectance and transmission measurements. The condenser cassegrain 18and the flat mirror 22 are used primarily for transmittancemeasurements. For reflectance measurements the coupling optic 24 istilted to direct the radiation to the transmittance/reflectance mirror14 which then directs a substantial part of the radiation down throughthe objective cassegrain 16 on to the sample. The radiation is reflectedfrom the sample back through the objective cassegrain 16. It is thereflectance mode with which embodiments of the present invention areconcerned. The apparatus also includes a detector and a cassegrainarrangement 26 which is used to carry out the spectroscopic analysis inconjunction with an IR spectrometer not shown. The operation of anarrangement of this type will be known to those skilled in the art andmore details of the operation of such an arrangement as used inconjunction with an ATR crystal can be found for example in EP-A-0730145and EP-A-0819932.

The present description is concerned with an accessory which can belocated on the movable stage 20 of a microscope to enable ATR imagingmeasurements to be carried out. Referring to FIGS. 2 to 9 of thedrawings an embodiment of the accessory comprises a support in the formof a baseplate 40 which is connected to a bracket 42 by means of screws43. The baseplate 40 locates in a recess 22 formed in the movable stage20 of the microscope. The stage 20 is movable in x, y and z directionsunder processor control by means of appropriate motors as will be knownto those skilled in the art. The baseplate 40 is held in position in therecess 22 by means of screws which are not shown in the drawings. Thebracket 42 provides a means of holding the accessory when locating it onor removing it from the stage 20.

The lower surface of the baseplate 40 is recessed at 44 and a bore 45extends through the baseplate and communicates with the recess 44.

The upper surface of the baseplate 40 supports an anvil 60 whichcomprises a sample supporting member. The anvil 60 is generally circularin plan and is located within the circular opening in a collar 62. Theouter edge of the collar, as can be seen in FIG. 2, is generally squareand the inner circular opening in the collar is slightly larger than theouter circular surface of the anvil so that the anvil can move to arestricted extent within the confines of the collar.

The anvil has a relatively thick annular side wall 63 and a relativelythin top wall 64. A recess 65 is defined between the top wall 64 and theside wall 63. The top surface of the anvil carries a removable plate 67,the top surface of the plate 65 constituting a sample supportingsurface. A projection 66 extends radially outwardly from the wall 62 tolocate beneath an overhanging part of the collar. This arrangementallows a small vertical movement of the anvil.

The anvil is held in place within the collar by means of two manuallyoperable adjusting screws 70, 71 located towards the front of the collarand a spring 74 (FIG. 4) disposed at the rear of the collar. The springis held in place by a screw 75. The screws 70, 71 are arranged so thattheir axes extend orthogonally towards the axis of the anvil and thespring 74 is designed to act along a bisector of the angle between theaxes of the screws. Thus, by manually operating the screws 70, 71 it ispossible to cause movement of the anvil within the confines of thecollar 62. Each screw is arranged to act against a surface on the anvil,which surface is inclined at a small angle to an axis through the anvil.The spring 74 is also arranged to act at a slight angle and thisarrangement ensures that there is a small vertical force imparted to theanvil which acts to press the anvil towards the baseplate 40.

The anvil arrangement described above constitutes a sub-stage which canbe moved relative to the electronically movable stage 20 of themicroscope itself.

A pressure exerting mechanism 80 for exerting pressure on the undersideof the top wall 64 is disposed below the anvil 60. The pressure exertingmechanism includes a tubular insert 81 which is threaded internally andwhich locates within the bore 45 of the baseplate. At its upper end, thethreaded insert 81 accommodates a ball bearing 82 which is disposed in aball guide 83. The ball bearing is biased upwardly into contact with theunderside of the top surface 64 by a spring 84 which pushes on the ballby way of a top-hat plunger 85. This plunger 85 is disposed in a plungerguide tube 86 threaded within the tubular insert 81 and extendingdownwardly into the recess 44. The spring is retained in the guide 86 bymeans of a screw 89 which can be used to adjust the pre-load of thespring.

A pinion gear 90 is fixed around the lower end of the guide tube 86 andconnected to the screw. The pinion gear 90 is coupled to a rack 91 byway of an idler gear 92 which can rotate about a spindle 93. The rackcan be moved longitudinally by means of a manually operable knob 94shown in FIG. 2. When the rack is moved longitudinally this causesrotation of the pinion gear 90 and this in turn causes a correspondingrotation of the spring plunger retaining guide tube within the tubularinsert 81. Depending upon the direction of movement of the rack,rotation of the retaining guide tube 86 raises or lowers the tube 86thereby either applying a lifting force through the ball 82 to the anvilor allowing the anvil to fall.

A mounting for an ATR crystal is carried above the anvil 60. Thismounting comprises an elongate arm 100 supported at its opposite ends.The arm has a first end 101 with an annular formation 102 within whichis located a pair of bushings and which locates over a guide pin 103carried on the baseplate 40. The arm also has a central portion with anaperture 105 which has stepped sides. The aperture comprises thelocation for an ATR crystal 106 which is shown in FIG. 4.

The arm has a second end 108 which is located at a level lower than thatof the first end. The second end 108 has an L-shaped slot 109 which canreceive a second guide pin 110 carried on the baseplate 40. Lockingscrews 111 are provided to lock the arm 100 in position on the guidepins.

The first end 101 of the arm 100 is provided with a braking mechanismwhich operates in conjunction with the guide pin 103. Referring to FIG.9 the annular formation 102 has a split internal bushing 150 with upperand lower parts 151 and 152. A rigid braking ring 153 is disposedbetween the bushing parts 151 and 152. The internal diameter of thebraking ring is slightly greater than the external diameter of the guidering 103. The ring 153 is usually biased into contact with the pin 103by a spring 155 which is held in position in the annular formation 102by a spring retainer and brake release stop 156. Diametrically oppositeto the spring there is provided a brake release button 158 which is heldcaptive in the annular formation 102. The radially inner end of thebutton 158 locates against the braking ring 153. When the button 158 ispressed the ring 153 is moved radially to a position where no part of itis in contact with the pin 103 thus releasing the braking effect. Themechanism 156 limits the extent to which the ring 153 can be moved.

Referring to FIG. 8 the annular formation 102 also includes an axiallyextending bore 160 which is spaced radially from the pin 103. The bore160, when the arm 100 is in the position shown in FIG. 2, receives anupstanding support pin 162 carried on the base 40. If the arm 100 israised along the axis of the pin 103 so that the bore 160 moves clear ofthe pin 162, the arm 100 can then be rotated about the guide pin 103away from the position shown in FIG. 2. The pin 162 can then contact theunderside of the arm 100 to hold it in its raised position. The arm 100can be rotated back to the position shown in FIG. 2 and allowed to lowerto its original position when the pin 162 and bore 160 are aligned. Thearrangement of pin 162 and bore 160 in conjunction with the location ofthe guide pin 110 in the slot 109 ensures correct and reproduciblelocation of the arm 100 and therefore correct and reproducible locationof the crystal 106 on the optical axis of the microscope.

The crystal 106 is generally hemispherical and is made from germanium.The lower surface is generally in the form of a shallow cone and has aflat central area 112 which constitutes a sample contacting area. Thecrystal 106 is bonded within a mounting ring 108 which is held withinthe opening 105 in the arm 100. The aim can include a sliding dust cover(not shown) to cover the crystal when not in use.

In use the crystal is positioned with respect to the microscope so thatthe centre of sample contacting area 112 is substantially at the focusof the microscope. Where an ATR crystal is to be used for ATR imagingoptimisation of the crystal design is important. When carrying out ATRimaging as distinct from simple transmission or reflectance imaging awider field of illumination is required. Furthermore, in the case of animaging system which involves scanning, the illumination may well benon-uniform across the field of view so that some compensation has to bemade for this if effective clear images are to be obtained. Whilst thiscan be achieved in software, optical modelling work carried out by theinventors has shown that the radius of curvature of the crystal affectsthe uniformity of the illumination. It has been found that an optimumradius exists for a given arrangement of illumination optics anddetector such that variations across a designated image area can beminimised whilst throughput is maintained. This technique has shown thata crystal radius of 6.75 mm is an optimum for the arrangement shown inthe drawings. This figure can be arrived at by using a Ray tracingtechnique in conjunction with appropriate assumptions which take intoaccount polarisation effects. It is found by operating this procedurethat for a small radius of curvature on the ATR optic the energyreceived from the centre of the sample area is high but the energyreceived from the other parts is relatively low. The image of the sampleis thus highly spatially non-uniform in brightness even if the sampleitself is spatially uniform. As the radius of curvature of the ATR opticis allowed to increase the uniformity of signal across the sample areaimproves to a point where the signals from the centre and the edges areapproximately equal. The uniformity of illumination defined in this waycan be used as a metric by which to select an optimum radius ofcurvature. If the radius of curvature is allowed to increase further thetotal energy received from all points of the sample area is typicallyseen to increase initially passing through a peak value at a certainradius of curvature and then falling away again to a very large radius.The radius at which the peak total energy occurs can be selected as thedesign optimum referred to above.

The mounting of the arm 100 on the guide pins 103, 110 in the mannershown in the drawings enables the arm 100 to be removed from the guidepins by raising it upwardly. The arm can then be inverted about itslongitudinal axis, and replaced on the guide pins so that the samplecontacting surface of the crystal is uppermost. This enables the samplecontacting surface of the crystal to be inspected using the visualinspection facility of the microscope to thereby allow its condition tobe assessed. The arrangement of guide pins 103, 110 also allows the armto be raised and then pivoted about pin 103 to allow the crystal to bemoved out of the optical path of the microscope. This can occur becausethe top of guide pin 110 is lower than the guide pin 103. In thisposition the arm 100 is held in its raised position by the pin 162. Theslot 109 allows accurate relocation of the arm 100 by locating the pin110 in the slot 109. The locking screws allow the arm to be locked at aselected height, for example with the crystal in contact with a sample.

Additionally, the crystal 106 is provided with a registration mark atthe apex of the hemispherical surface. This mark can take the form of aflat formed on the hemispherical surface or some other faun of markingon the surface itself. This registration mark is used to correctlylocate the crystal both horizontally and vertically as will bedescribed.

The registration mark does not have to be at the apex of thehemispherical surface although this is the most convenient and preferredposition. The mark can be at any location on the accessory provided thatthat location is fixed mechanically relative to the sample contractingarea 12 and is visible through the viewing system of the microscope.

As can be seen from the drawings the arm 100 when it is located on thebaseplate 40 needs to be lowered towards the sample receiving surface.It is important that this movement be controlled in order to avoiddamage particularly to the crystal and to this end the accessory isprovided with the brake mechanism 153, 155, 156, 158 located within thestructure 120 which includes the guide pin 103. The brake mechanismnormally prevents the arm from dropping under gravity and is provided toavoid inadvertent damage to the crystal 106. The button 158 is operatedmanually to release the brake and allow the arm 100 to be lowered. Theuse of a releasable braking mechanism enables a user to lower the armwithout any friction between the arm and guide pin and therefor givesthe user greater control of the lowering operation.

The registration mark provided preferably at the apex of the crystal 106is used to align the crystal with the optical axis of the microscopethereby providing a defined starting position for any imaging scan. Thesoftware of the instrument is provided with lateral offset parameterswhich permit an ATR image to be aligned precisely with the visible lightimage or with a conventional transmission/reflectance image. Also thesoftware is provided with a precalibrated crystal height parameter whichdefines the distance by which the crystal 106 and sample should beraised from an initial position in order to bring the sample surfaceinto sharp focus via the infrared part of the microscope system. This issignificant because the crystal 106 is opaque to visible light andmanual sample focusing is not possible when the crystal is in place.

In order to register the crystal position, the crystal 106 and itsmounting arm 100 are located above the sub-stage or anvil with no samplein position. The user of the instrument is prompted to focus using thevisible camera on a point on the arm 100 known as the starting point.This is illustrated at 120 in FIG. 10 of the drawings. The system thenoperates to move by a predetermined distance in an x, y z co-ordinatesystem to the centre of the top of the crystal which is fiducial orregistration mark. The user then confirms that the centring and focusare correct making any small adjustments that may be required. Thesystem then sets the stage co-ordinate system origin (0,0,0) as theconfirmed position.

The calibration of the crystal height and focus setting can be carriedout as follows. In this respect it needs to be appreciated that smallvariations in the crystal shape (radius of curvature and thickness) cangive rise to significant shifts in the required focus setting. As therefractive index of germanium is high, focus cannot be establishedvisually because the crystal is opaque to visible light and it requiresa sample with a well defined spatial structure as well as a strongspectral absorption band that can be detected by ATR. A novel method hasbeen developed by the inventors whereby the optimum ATR focus settingcan be determined. This method makes use of a test sample whichpreferably comprises a small section of 3m Vikuiti BEF 11 film. This isa plastic sheet the surface of which is micro embossed with a set ofparallel triangular prisms each of which has a 90° apex angle and aperiod of 50 or 24 microns. The material is commonly employed to improvethe brightness of LCD display panels. The ATR crystal is brought intocontact with the prism structures so that the apices are flattened bythe contact pressure thereby resulting in a set of parallel rectangularcontact zones with the same pitch as the basic material. Each zone hassharp edges. In order to determine the optimum focus small fineresolution images are acquired around the centre of the field and narrowwaveband images are extracted which are centred on the material'sspectral absorption. The technique involves extracting absorption imagecross sections orthogonal to the edges. The degree of focus is estimatedby inspecting the slopes of the cross sections across the contact edges.Measurements are taken at various vertical positions of the crystal andthe slopes increase as optimum focus is approached and drop away againwhen departing from the optimum focus. The best position can be found byinterpolating between a set of scans taken at different focus settings.It should be noted that the deformation of the sample film depends uponthe contact pressure and this can be used as a means of confirming thatadequate pressure is being applied to the sample.

Thus in more general terms crystal calibration is achieved by using atest sample with particular properties and examining infra-red images ofthis sample for spatial sharpness. The vertical displacement of thecrystal is varied in steps and image sharpness is recorded at each stepin order to determine an optimum displacement. This involves

-   -   a) An infra-red image is acquired at an initial vertical        displacement which is estimated by finding the position at which        maximum infra-red energy is transmitted through the crystal.        This may not be the same as the best focus position.    -   b) The image is spectrally filtered to extract a spatial map of        the absorbance at a wavelength where the sample absorbs        strongly. This is a function of the material properties.    -   c) A cross-section of the absorbance map at this wavelength is        extracted which traverses spatially sharp features in the        image—in the present case an edge between a polymer and air.    -   d) The cross-section is differentiated mathematically to extract        slope information, and the maximum slope is measured for a        recognisable feature in the image.    -   e) The crystal displacement is adjusted iteratively so as to        maximise the slope of the cross-section at the given feature.        The vertical crystal position where maximum slope is obtained is        the position at which the image of the sample is in best focus.        This value (measured with respect to the index mark) is recorded        as the crystal height parameter which is supplied to a user.    -   f) Any new/replacement crystal will have a new calibration value        which a user must input to the control software of the        instruments.

In principle the test sample can be any material with infra-redabsorption which can provide spatially sharp features which are ideallysmall compared to the anticipated spatial resolution of the microscopesystem. This means typically features which are sharp on a scale ofabout 3 microns.

The test sample should be “ATR-compatible”—in other words it shouldprovide a clear spectral absorption within the spectral range of the ATRaccessory and the sharp features must survive being pressed intointimate contact with the sample surface without becoming smeared out orsmoothed.

The sharp features might include engineered fine structures such aslines, gratings or grids, or alternatively sharp edges between twodifferent materials (one of which might be air in the form of a void).If the features are constructed by forming indentations in a uniformmaterial, then the indentations should have a depth of more than a fewmicrons when in contact with the crystal, and the transitions fromcontact to non-contact should be sharp.

As explained above the presently preferred material is a particularmicroembossed polymer sample (Vikuiti light control film manufactured by3M) because it provides a convenient, cheap and reproducible testsample. The material comprises a regular array of roof prisms whoseridges are conveniently flattened by contact with the crystal to leavestraight “bars” of contact. The geometry of the edges of these contactregions is such that the material drops away from the crystal rapidly,especially over the first few microns of depth, and this yields a veryhigh quality edge between absorbing polymer and non-absorbing air whichis easily imaged using ATR to give high-contrast and sharp results.

The inventors have also developed a technique for definition of spatialresolution. The technique described above for determining the optimumfocus of the crystal can be adapted to provide a measurement of theeffective resolution of the ATR system in a manner which is difficult toachieve on systems which employ staring two dimensional array detectors.Having determined an optimum focus setting as described above, a smallstrip image is acquired across one or more of the edges in the testtarget but using a very fine step in the direction across the edges.This results in an image which is over-sampled in this direction. Across section is extracted in the absorption band of the sample anddifferentiated by means of a digital filter. The profile thus obtainedapproximates to a cross section through the point spread function of theoptical system and can be used to estimate the resolving power of thesystem either directly or via appropriate curve fitting.

In order to carry out measurement of a spectrum on a sample the firststep is to register the crystal position and define the stageco-ordinate system origin (0,0,0) as described above with reference toFIG. 10. The next step is to measure the background spectrum that is tosay without a sample in position on the plate 67. In order to achievethis the user sets the mounting arm 100 to an arbitrary position on theguide pins 110 and 103 such that the sample contacting surface of thecrystal is spaced from the plate 67 to thereby provide a crystal/airinterface. The user then causes the stage to move to the origin, that isto say the top centre of the crystal and to fine focus on top of thecrystal. The user can then select the resolution and pixel size. Thesystem then operates automatically to raise the stage 20 by apredetermined distance based on the stored crystal height parameter inorder to focus on the lower surface of the crystal (106). The systemthen carries out a measurement on the background spectrum for eachdetector in the array and these are stored. The way in which thebackground spectrum is obtained will be apparent to those skilled in theart.

The next step is to measure the crystal image and this involves the usermoving the stage to the origin, i.e. the top centre of the crystal. Theuser fine focuses on the top of the crystal and then enters parameterssuch as the resolution, scans per pixel, pixel size and image size. Thesystem raises the main stage 20 by a predetermined distance in order tofocus on the lower surface of the crystal. The system measures the imageof the crystal without a sample being present and this is stored.

The next step is to measure a sample and the first step is for a user toselect an optional processing option which is subtraction of thebackground crystal image or baseline offset correction. The user thenraises the arm 100 and pivots it about the pin 103 so that the crystal106 is moved away from the optical axis of the microscope. The plate 67on the anvil 60 is then removed and a sample located on that plate andthe plate placed back in position on the anvil 60. The system thenraises the stage by a predetermined distance in order to enable visualinspection of the sample using the visual facility of the microscope.The user mounts the sample in four sub-steps. These are:

(i) Mount the sample on the plate 67 and locate on the anvil 60.

(ii) Adjust the sample position using the sub-stage only, i.e. using theadjusting screws 70, 71 to adjust the position of the anvil (60). Thisis carried out whilst viewing the sample visually.

(iii) Swing the arm 100 back into position so that the crystal 106 islocated on the optical axis of the microscope and lower the arm 100thereby lowering the crystal 106 into contact with the sample on theplate 67.

(iv) Apply pressure to the underside of the top of the anvil 60 usingthe pressure applying mechanism 80 so that the sample is pressed intocontact with the crystal.

With regard to (iv) ATR measurement requires that a sample is held ingood contact with the lower face 112 of the crystal 106. Ideally thecontact pressure should be reasonably uniform across the face of thecrystal. For hard samples it is necessary for the sample to lie parallelto the crystal face; for most compliant samples control of the clampingforce is required to prevent excessive sample deformation. With thesample on the plate 67 the arm 100 is lowered until the crystal justcontacts the sample. The arm is locked using screws 111. The rack 91 isoperated to apply a lifting force to the anvil 60 thus compressing thesample against the crystal face. This force is produced through thespring plunger 85 which is designed to restrict the maximum force to avalue which avoids damage to the crystal 106. The force vector isimportant. Ideally it should be aligned with the centre of the crystalface so that samples tend to align to the face and contact pressure isuniform. This is achieved by use of the ball bearing 82 that isaccurately constrained in its guide 83 and precisely aligned with thecrystal axis. The ball acts against a thin part of the anvil so that thelifting force originates as close as possible to the crystal. Thearrangement of the rack, pinion, spring plunger, lifting spring and ballis very compact in height and therefore significant in a microscopewhere there is limited space. The design of the spring plunger 85ensures that the lifting spring 84 within the assembly is nevercompressed sufficiently to become coil bound and thus exert excessiveforce.

The screw 89 can be used to vary the pre-load on the spring. The maximumtravel for the screw is set such that the spring never becomescoil-bound. With the screw set full clockwise the pre-load isapproximately 50% of the maximum spring force. When the compressioncontrol knob is moved to the maximum pressure position the spring forceincreases close to the maximum rating of the spring. Backing off thescrew 89 by one turn reduces the pre-load to zero. Thus with control atone end of its travel no force is imparted to the sample. As the userslides the control knob the compressive force gradually increases to 50%of the maximum possible spring force.

After sub-step (iv) the system then lowers the stage 20 by apredetermined distance in order to focus on the crystal top. The userfine focuses the registration mark on the top of the crystal ifnecessary. The user then enters the resolution, scans per pixel, wavenumber range, pixel size and image size and after this the system raisesthe stage 20 by a predetermined distance based on the stored crystalheight parameter to focus on the sample. A measurement of the sampleimage is then taken in a manner which will be apparent to those skilledin the art. Where imaging is being carried out this involves making afirst measurement, moving the microscope stage slightly to carry out asecond measurement and repeating this for different positions on themicroscope stage. Finally the system performs the selectedpost-processing and stores the image.

It should be understood that the motorised stage 20 is used for aligningthe crystal 106 directly under the centre of the microscope field andfor scanning the image around this centre. To align the sample the usershould not use this motorised stage and this is the reason for theprovision of the sub-stage 60. The software procedure can disable themoving mechanism of the motorised stage during sample viewing so that auser cannot inadvertently change the crystal registration.

The arrangement is provided with a rapid automated spectral analysisprocedure which is illustrated in FIG. 11 of the drawings. In manypractical cases the sample presented for ATR analysis may haverelatively weak overall absorption. ATR images showing total absorptionobtained from such samples are often somewhat featureless or of very lowcontrast and illumination artifacts may obscure the details of thesample. Compensation for illumination effects is possible usingconventional means such as ratios against background images or baselinecorrection which normalises spectra using regions known to haveessentially zero absorption. It is possible also to process the datausing advanced techniques in order to extract and display significantspectral features but this requires expertise in spectral signalprocessing and can be time consuming. In order to provide the user ofthe present system with a quick indication of the spectral informationpresent in the image an automatic processing sequence has been devised.Its aim is to extract the most significant spectral features from theraw data in a manner which is independent of the sample and to permitthe operator to display these either singly or in the form of colourcomposite images. The benefit to the user is the rapid confirmation thatthe image contains potentially useful information and a good preliminaryanalysis that provides guidance for further more advanced processing.For example the results can indicate which parts of the image arespectrally similar and which are distinct allowing the user to make asimple segmentation of the sample. The raw image may be acquired witheither ratiometric correction against the background or baseline offsetcorrection. The processing sequence begins with a step designed tominimise the effects of offsets and baseline curvature which is shown at130 in FIG. 11. This involves differentiation in the spectral domain(first derivative with some degree of smoothing to reduce noise) andsubtraction of any remaining average value. The spectral image is thenrestricted as shown at 131. At the short wave side a limit is settypically at about 3300 wave numbers since few samples show significantabsorptions at shorter wavelengths. A small restriction is also made atthe longwave end of the range to improve overall signal to noise. Theaverage absorbance is then subtracted as shown at 132 and a principalcomponents analysis is then applied as shown at 134 in order to extractthe most significant spectral features. The components can then bepresented to the user in an interface which allows them to be displayedas colour composite images as shown at 136.

The invention claimed is:
 1. A method of carrying out automatedattenuated total reflectance (ATR) spectral measurement, the methodcomprising the steps of: differentiating an acquired image in a spectraldomain; restricting a spectral range of the acquired image; subtractingan average absorbance from the restricted image data; and applying aprincipal component analysis to extract significant spectral featuresfrom the restricted image data.
 2. The method of claim 1, furthercomprising color compositing the extracted features for display to auser.
 3. The method of claim 1, wherein the differentiating stepcomprises smoothing the acquired image to reduce noise.
 4. A method ofcarrying out automated attenuated total reflectance (ATR) spectralanalysis, the method comprising the steps of: acquiring a spatial imageof a sample; differentiating the acquired image in a spectral domain;restricting a spectral range of the image; subtracting an averageabsorbance from the restricted image data; applying a principalcomponents analysis to extract the most significant spectral features;and displaying a spatial image showing the most significant spectralfeatures of the sample to a user.
 5. The method of claim 4, furthercomprising color compositing the extracted components for display to theuser.
 6. The method of claim 4, wherein the differentiation stepincludes smoothing the acquired image to reduce noise.
 7. An imagingattenuated total reflectance (ATR) system, comprising: an imagingmicroscope; image processing means; and an interface for displayingprocessed images to a user, wherein the system is constructed andarranged to carry out automated ATR spectral analysis by: acquiring animage of a sample; differentiating a acquired image in a spectraldomain; restricting a spectral range of the image; subtracting anaverage absorbance from the restricted image data; applying a principalcomponents analysis to extract the most significant spectral features;and displaying a spatial image showing the most significant spectralfeatures via the interface.
 8. The imaging ATR system of claim 7,wherein the system is further arranged for color compositing theextracted components for display to the user.
 9. The imaging ATR systemof claim 7, wherein the differentiation step includes smoothing theacquired image to reduce noise.